UNIVERSITY   OF  CALIFORNIA   PUBLICATIONS 

IN 

AGRICULTURAL    SCIENCES 

Vol.  1,  No.  6,  pp.  127-139  March  28,  1914 


THE  EFFECT  OF  COPPER,  ZINC,  IRON  AND 

LEAD   SALTS  ON  AMMONIFICATION 

AND  NITRIFICATION  IN  SOILS 

BY 

C.  B.  LIPMAN  and  P.  S.  BUEGESS 


In  the  course  of  investigations  on  the  effect  of  smelter  wastes 
on  crop  growth  one  of  the  writers  decided  to  test  also  the  effects 
of  the  salts  of  the  heavy  metals  on  the  transformation  of  organic 
nitrogen  in  the  soil  into  ammonia  and  nitrates  which  serve  as 
the  source  of  nitrogen  for  plants.  This  correlation  between  the 
effects  of  external  factors  upon  both  the  soil  flora  and  the  physio- 
logical condition  of  plants  was  deemed  eminently  worth  while, 
for  reasons  which  are  at  once  obvious  to  the  careful  student  of 
soil  fertility  problems.  Some  of  the  results  obtained  with  plants 
have  been  published,1  and  still  fuller  data  dealing  with  the  same 
problem  will  soon  find  publication  elsewhere.  In  view  of  the 
foregoing  the  writers  have  carried  out  experiments  dealing  with 
the  toxic  and  stimulating  effects  of  copper,  zinc,  iron  and  lead 
on  the  ammonifying  and  nitrifying  flora  of  a  sandy  soil.  The 
results,  owing  to  their  interesting  nature  and  their  cogency  at 
this  time  are  here  given  apart  from  the  results  obtained  with 
plants. 

Methods  of  the  Experiments 

The  work  here  described  was  carried  out  by  the  direct  soil- 
culture  method  in  a  manner  fully  explained  by  one  of  us  else- 
where.2   No  attempt  was  made  to  modify  the  soil  flora,  but  the 


i  Bot.  Gaz.,  vol.  55,  p.  409. 

2  Cent,  fiir  Bakt.,  2te  Abt.,  vol.  32,  p.  58,  vol.  33,  p.  305. 


128         University  of  California  Publications  in  Agricultural  Sciences     [Vol.  1 

soil  with  its  natural  flora  was  employed.  The  ammonia  deter- 
minations were  made  in  accordance  with  methods  described  in 
the  papers  above  cited  and  the  nitrate  determinations  in  accord- 
ance with  the  method  described  by  Burgess.3 

The  work  carried  out  by  other  investigators  dealing  with  the 
subject  in  hand  has  thus  far  been  rather  meager  and  but  few 
of  the  results  possess  pertinence  with  respect  to  our  findings.  In 
studying  the  amounts  of  copper  in  vineyard  soils  due  to  accumu- 
lation from  copper-containing  sprays,  Prandi4  was  unable  to 
note  any  damage  to  vines  after  the  copper  sprays  had  been  used 
for  a  number  of  years.  Moreover,  he  did  not  adjudge  dangerous 
the  amounts  of  copper  which  had  accumulated  in  the  soils 
studied  in  the  quantities  in  which  he  found  it  there.  He  does, 
however,  make  the  further  interesting  speculative  statement  that 
copper  may  have  an  important  influence  on  the  soil  organisms. 
The  original  work  of  Eickmeyer5  is  unfortunately  not  accessible 
to  us  and,  while  it  may  contain  some  of  the  most  cogent  infor- 
mation  on  our  subject  which  is  in  print,  we  can  ascertain  only 
that  the  investigator  named  studied  the  effects  of  copper  and  iron 
sulfates,  among  other  poisons,  on  soil  bacteria.  In  studying  the 
bacteriology  of  ammonium  compounds  Ehrenberg6  observes  that 
the  cause  of  the  difference  in  the  effects  of  ammonium  compounds 
used  for  fertilizers  in  zinc  pots  and  in  the  field  must  be  that  the 
zinc  exercises  a  deleterious  effect  on  "ammonia-fixing"  bacteria 
which  compete  with  the  plants  for  soluble  nitrogen  in  the  soil. 
Owing  to  that,  he  believes  ammonium  fertilizers  stimulate  plants 
in  zinc  pots  but  have  no  effect  in  the  field.  Among  studies  on 
the  effects  of  the  poisonous  metals  here  considered,  on  some  of  the 
lower  organisms,  may  be  mentioned  those  of  Latham,7  who 
obtained  stimulation  with  low  concentrations  of  ZnS04  for 
Aspergillus  nigcr,  and  those  of  Remy  and  Rosing,8  which  indicate 
a  marked  stimulating  effect  of  iron  on  Azotobacter  organisms. 


3  Univ.  Calif.  Publ.  Agr.  Sci.,  vol.  1,  no.  4,  p.  51. 

'  8taz.  Sper.  Gr.  Ital.,  vol.  40,  p.  531,  cited  from  E.  S.  R.,  vol.  19,  p.  755. 

•-•  Winner  Landw.  Ztg.,  vol.  57,  p.  600,  cited  from  E.  S.  R.,  vol.  19,  p.  625. 

8  Fuhling's  Landw.  Zt<>'.,  vol.  27,  p.  449. 

7  Cited  from   E.  S.  R.,  vol.  21,  p.  421. 

-  Ciit.  fiir.  Bakt.,  2te  Abt.,  vol.  30,  p.  349. 


1914]       Lipman-Burgess:  Ammonification  and  Nitrification  in  Soils  129 

The  only  work  with  which  the  writers  are  acquainted  which 
allows  of  any  direct  comparison  with  our  results  is  included  in 
the  splendid  series  of  investigations  of  Fred9  on  the  effects  of 
small  or  limited  quantities  of  various  poisons  on  the  higher  as 
well  as  the  lower  plant  organisms.     In  addition  to  showing  the 

p  u    \ 

stimulating  powers  of  certain  poisons  like  CS2  and  p2Tx '  )  0 

on  the  bacterial  count,  nitrogen-fixing  power,  etc.,  of  soils  in  direct 
soil  cultures,  that  investigator  gives  evidence  also  of  work  done 
with  the  higher  plants  yielding  similar  results.  Of  a  series  of 
metallic  salts,  further,  with  which  Fred  worked  he  was  unable  to 
demonstrate  stimulation  to  nitrification  except  perhaps  one  case 
of  slight  stimulation  through  the  use  of  FeS04  (100  mgs.  per  100 
grams  of  soil).  The  salts  tested  were  MnS04,  FeS04,  CuS04, 
and  NaCl.  The  nitrifiable  material  employed  was  (NH4)2S04 
and  not  the  organic  nitrogen  of  blood  which  we  used. 

Aside  from  these  investigations,  only  one  or  two  of  which 
have  any  direct  bearing  on  our  subject,  but  little  has  been 
accomplished  of  either  direct  or  indirect  applicability  to  our 
results.  Where  any  studies  were  carried  out  dealing  with  the 
effects  of  the  heavy  metals  on  bacteria,  such  as  those  of  Kellerman 
and  Beckwith10  or  Jackson,11  they  were  prosecuted  in  solutions 
and  therefore,  as  one  of  us  has  emphatically  shown  in  other 
publications,12  are  scarcely  comparable  with  results  from  soil  cul- 
tures such  as  are  below  discussed,  or  with  results  obtained  with 
plants  in  soil  cultures. 

Since  the  experiments  below  described  were  completed  there 
has  appeared  from  the  pen  of  Greaves13  a  paper  bearing  on  the 
effects  of  arsenic  on  ammonification  and  nitrification  in  soils.  This 
work,  because  of  the  methods  employed,  is  of  pertinence  here, 
and  interesting,  because  of  the  stimulating  powers  of  considerable 
quantities  of  arsenic  in  soils  to  ammonia  and  nitrate  production 
by  the  natural  flora.     While  arsenic,  chemically  considered,  is  a 


9  Cent,  fur  Bakt.,  2te  Abt.,  vol.  31,  p.  185. 
io  Bull.  100,  Bur.  PI.  Ind.,  U.  S.  D.  A. 
11  Jour.  Am.  Chem.  Soc,  vol.  27,  II,  p.  675. 
12Bot.  Gaz.,  vol.  55,  p.  409  and  literature  there  cited. 
13  Cent,  fur  Bakt.,  2te  Abt.,  vol.  39,  p.  542. 


130         University  of  California  Publications  in  Agricultural  Sciences     [Vol.  1 

totally  different  substance  from  the  metals  we  have  studied,  it 
is  interesting  to  compare  its  effects  on  the  soil  flora  with  those 
exercised  by  copper,  zinc,  lead  and  iron.  This  is  especially  so 
since  arsenic  so  frequently  occurs  with  the  other  elements  in 
soils  and  in  smelter  wastes  from  which  it  may  be  transported  to 
agricultural  soils. 

In  closing  this  brief  review  of  investigations  dealing  with  the 
subject  of  this  paper  or  one  allied  thereto,  it  should  also  be 
added  that  some  fragmentary  information  was  obtained  in  the 
course  of  other  investigations  by  J.  G.  Lipman  and  his  asso- 
ciates14 which  refers  to  the  effects  of  CuS04,  and  FeS04  among 
other  compounds  on  the  ammonifying  flora  of  the  soil.  These 
results  indicate  that  copper  stimulates  ammonification  very 
slightly  at  a  concentration  of  0.1  per  cent,  that  ZnS04  gives  no 
stimulation  and  is  toxic  at  all  concentrations,  and  that  FeS04 
at  the  concentration  of  1  mg.  per  100  grams  of  soil  gives  marked 
stimulation. 

The  Ammonification  Experiments 
To  a  fifty-gram  portion  of  dry  soil,  one  gram  of  tankage 
(9.62%  N)  was  added  and  thoroughly  mixed  with  it.  Sterile 
distilled  water  was  then  added,  to  make  optimum  moisture  con- 
ditions, and  also  varying  quantities  of  the  salts  to  be  tested. 
The  soil,  water  and  salt  mixture  was  thoroughly  stirred  in  the 
tumbler,  the  latter  covered  with  a  Petri  dish  cover  and  incubated 
for  one  week  at  27°  C  to  30°  C.  The  results  of  the  ammonia 
determinations  at  the  end  of  the  incubation  period,  together  with 
other  necessary  explanatory  data,  will  be  found  in  Table  1.  The 
sulfates  of  the  metals  tested  were  employed  throughout.  It  will 
be  noted  that  the  results  of  duplicate  determinations  are  given 
throughout,  averages  being  omitted  since  the  reader  can  so 
readily  ascertain  them  from  the  data  submitted. 

It  is  at  once  obvious,  from  even  a  casual  examination  of  the 
data  given  in  Table  1,  that  none  of  the  metals  employed  exercises 
a  stimulating  effect  on  the  ammonifying  flora  of  the  soil  employed 
even  at  the  smallest  concentrations   (50  p.p.m.).     Despite  that 


"  N.  .1.  Exp.  Sta.  Bull.  no.  246,  p.  32. 


1914]       Lipman-Burgess:  Ammonification  and  Nitrification  in  Soils  131 


TABLE 

1 

The  Effect  of  Cu 

,  Zn,  Fe 

,  AND  PB 

on  Ammonification 

IN  Soils 

No. 

1 

CuS04 

i  of 

dry  soil 
0 

N  as  NH:j 

produced 

mgs. 

40.60 

ZnS04 

to  Of 

dry  soil 
0 

N  as  NH.3 

produced 

mgs. 

40.60 

FeS04 

a  of 

dry  soil 
0 

N  as  NH3 

produced 

mgs. 

40.60 

PbS04 

*  of 

dry  soil 

0 

N  as  NH3 

produced 

mgs. 

40.60 

2 

0 

39.20 

0 

39.20 

0 

39.20 

0 

39.20 

3 

.005 

40.32 

.005 

36.82 

.005 

39.34 

.005 

38.50 

4 

.005 

38.50 

.005 

36.68 

.005 

37.94 

.005 

37.94 

5 

.010 

36.40 

.010 

39.20 

.010 

39.48 

.010 

37.52 

6 

.010 

lost 

.010 

37.38 

.010 

37.38 

.010 

38.78 

7 

.025 

35.00 

.025 

36.82 

.025 

37.24 

.025 

41.58 

8 

.025 

35.70 

.025 

35.98 

.025 

lost 

.025 

39.90 

9 

050 

35.98 

.050 

33.74 

.050 

37.10 

.050 

40.32 

10 

.050 

36.26 

.050 

32.20 

.050 

39.20 

.050 

38.78 

11 

.075 

32.20 

.075 

31.50 

.075 

36.68 

.075 

39.76 

12 

.075 

30.80 

.075 

31.92 

.075 

37.94 

.075 

38.92 

13 

.100 

29.96 

.100 

31.08 

.100 

38.22 

.100 

35.70 

14 

.100 

28.00 

.100 

29.12 

.100 

39.34 

.100 

35.84 

15 

.125 

27.58 

.125 

31.50 

.125 

35.98 

.125 

35.00 

16 

.125 

28.84 

.125 

31.50 

.125 

34.58 

.125 

35.42 

17 

.150 

28.70 

.150 

34.30 

.150 

35.56 

.150 

33.04 

18 

.150 

29.96 

.150 

33.74 

.150 

37.52 

.150 

34.72 

19 

.200 

26.66 

.200 

34.58 

.200 

36.40 

.200 

35.00 

20 

.200 

27.44 

.200 

33.60 

.200 

37.10 

.200 

32.90 

21 

.250 

28.70 

.250 

32.90 

.250 

36.68 

.250 

35.00 

22 

.250 

27.58 

.250 

32.06 

.250 

37.52 

.250 

36.26 

fact,  however,  the  toxicity  of  no  one  of  them  is  very  marked- 
While  this  latter  observation  is  in  accord  with  the  findings  of 
J.  G.  Lipman  above  referred  to,  the  former  result  is  entirely  at 
variance  with  them.  It  is  significant  to  remark  in  this  connec- 
tion, however,  that  the  agreement  between  duplicate  determina- 
tions was  on  the  whole  much  better  in  our  experiments  than  in 
those  carried  out  at  the  New  Jersey  Experiment  Station  and 
should  therefore  render  our  results  more  trustworthy.  Indeed, 
as  the  authors  of  the  New  Jersey  bulletin  state,  for  their  work 
"there  is  not  sufficient  evidence  upon  which  to  base  conclusions." 
Comparing  the  relative  toxicities  of  the  salts  tested  by  us  it 
would  appear  that  copper  is  the  most  toxic,  zinc  the  next,  lead 
the  next,  and  iron  the  least  toxic  of  the  four  sulfates.  This 
assertion  is  based,  on  the  fact  that  at  the  highest  concentrations 
at  which  it  was  used,  copper  depresses  ammonification  by  about 


132         University  of  California  Publications  in  Agricultural  Sciences     [Vol.  1 

30  per  cent,  zinc  by  20  per  cent,  lead  by  about  15  per  cent,  and 
iron  by  only  about  12  per  cent  of  that  of  the  normal  yield. 
Despite  this  fact,  however,  copper  begins  to  manifest  its  toxicity 
most  slowly  of  the  four  metals,  since  it  shows  little  if  any  toxicity 
at  a  concentration  of  .005  per  cent,  whereas  all  the  other  metals 
show  clearly  a  slight  toxic  effect  even  at  that  concentration. 
Just  why  this  peculiar  relationship  between  the  metals  tested 
and  the  ammonifying  flora  should  obtain  is  by  no  means  easy 
of  explanation. 

Speaking  of  the  data  in  Table  1,  by  and  large,  it  seems  justi- 
fiable to  venture  the  assertion  that  all  the  metals  become  markedly 
more  toxic  at  concentrations  of  0.1  per  cent  of  the  dry  weight 
of  the  soil  or  in  concentrations  in  excess  of  that  amount.  Other 
details  worthy  of  mention  here  are  the  fact  that  at  times  the 
depressing  effect  of  a  certain  concentration  is  followed  by  a 
stimulating  effect  at  an  increased  concentration  of  the  same 
metallic  salt.  Also,  the  very  striking  fact  is  noted  of  the  rela- 
tively slight  increases  of  toxicity  which  accompany  relatively 
large  increases  in  concentration  of  the  salt.  The  latter  is  par- 
ticularly marked  in  the  case  of  FeS04.  Whether  or  not  this 
latter  result  is  to  be  explained  by  the  differences  in  solubility 
which  obtain  between  the  different  metallic  sulfates  employed,  as 
well  as  by  the  different  adsorptive  power  exercised  by  the  soil 
towards  every  one  of  them,  still  remains  to  be  shown. 

In  general,  it  is  worthy  of  special  remark  here  that  though 
these  so-called  very  toxic  metals  manifest  undoubted  toxicity 
towards  the  ammonifying  flora,  and  no  stimulation  in  any  con- 
centration of  them,  the  toxic  effect  is  relatively  small  and  in 
some  cases  even  absolutely  slight. 


The  Nitrification  Experiments 

To  100-gram  portions  of  sandy  soil  in  tumblers  were  added 
two  grams  of  dried  blood  (13.29%  N),  enough  water  to  make 
optimum  moisture  conditions,  and  the  necessary  amounts  of  the 
salts  to  be  tested.  The  mixture  was  prepared  for  incubation  as 
above  described  for  the  amm on ifi cation  cultures  and  incubated 


1914]       Lipman-Burgess :  Ammonification  and  Nitrification  in  Soils  133 

for  four  weeks  at  27°  C  to  30°  C.  Other  explanatory  data  are 
given  in  Table  2,  which  shows  the  results  obtained  from  the 
nitrate  determinations  made  as  above  explained. 


TABLE  2 

The  Effect  of 

Cu,  Zn,  Fe,  and  Pb 

on  Nitrification 

in  Soils 

No. 
1 

CuS04 

dry  soil 
0 

Nitrates 

produced 

mgs. 

10.64 

ZnS04 

*  of 

dry  soil 

0 

Nitrates 

produced 

mgs. 

11.20 

FeS04 

*  of 

dry  soil 

0 

Nitrates 

produced 

mgs. 

11.20 

PbS04 

i  of 

dry  soil 
0 

Nitrates 

produced 

mgs. 

11.20 

2 

0 

9.80 

0 

10.64 

0 

10.64 

0 

10.64 

3 

.0125 

7.84 

.0125 

10.36 

.0125 

14.56 

.0125 

5.60 

4 

.0125 

10.64 

.0125 

11.48 

.0125 

16.24 

.0125 

7.00 

5 

.0250 

11.20 

.0250 

11.20 

.0250 

12.60 

.0250 

9.52 

6 

.0250 

10.08 

.0250 

12.60 

.0250 

12.88 

.0250 

8.68 

7 

.0500 

17.64 

.0500 

16.24 

.0500 

13.72 

.0500 

10.08 

8 

.0500 

18.76 

.0500 

17.36 

.0500 

13.44 

.0500 

11.20 

9 

.0750 

24.36 

.0750 

21.00 

.0750 

14.00 

.0750 

11.48 

10 

.0750 

24.92 

.0750 

23.80 

.0750 

11.76 

.0750 

14.00 

11 

.1000 

23.80 

.1000 

23.52 

.1000 

8.40 

.1000 

7.56 

12 

.1000 

24.64 

.1000 

22.68 

.1000 

7.84 

.1000 

7.56 

13 

.1250 

21.00 

.1250 

17.08 

.1250 

18.20 

.1250 

11.20 

14 

.1250 

19.32 

.1250 

17.22 

.1250 

21.00 

.1250 

12.88 

15 

.1500 

20.44 

.1500 

17.08 

.1500 

20.72 

.1500 

6.44 

16 

.1500 

19.88 

.1500 

17.36 

.1500 

19.60 

.1500 

6.72 

It  is  quite  clear  from  the  foregoing  table  that  all  of  the  salts 
used  exercise  marked  effects  on  the  nitrifying  flora  of  the  soil 
employed.  For  the  reason  that  some  pronounced  differences 
obtain  between  the  action  of  the  different  salts  we  shall  discuss 
each  of  them  separately  at  first. 

CuS04 

It  can  be  seen  at  a  glance  that  the  data  in  Table  2  show 
copper  to  be  the  most  stimulating  of  the  salts  employed  so  far 
as  a  soil-nitrifying  flora  is  concerned.  While,  unfortunately, 
the  duplicate  determinations  for  the  lowest  concentration  do  not 
agree  well,  they  indicate  clearly  that  in  those  quantities  (0.0125 
per  cent,  or  125  parts  per  million)  copper  has  either  no  effect 
on  the  nitrifying  flora  or  a  slightly  depressing  one.  By  doubling 
the  concentration  of  the  copper  salt  we  obtain  a  slightly  stimu- 
lating effect,  but  by  doubling  the  latter  concentration  we  obtain 


134         University  of  California  Publications  in  Agricultural  Sciences     [Vol.  1 

a  most  striking  stimulating  effect  on  the  nitrifying  flora,  which 
results  in  the  production  of  nearly  twice  as  much  nitrate  nitrogen 
as  is  yielded  in  the  normal  soil  receiving  no  copper  at  all.  But 
the  stimulation  of  the  nitrifying  flora  does  not  cease  at  a  con- 
centration of  CuS04  equivalent  to  0.05  per  cent  of  the  dry 
weight  of  the  soil.  Even  at  a  concentration  of  0.075  per  cent  of 
CuS04  we  obtain  stimulation  and  one-third  again  as  much  nitrate 
as  is  produced  when  copper  is  present  at  the  preceding  concen- 
tration (0.05  per  cent  CuS04).  At  0.1  per  cent  CuS04  about 
the  same  amount  of  nitrate  is  produced  as  at  a  concentration  of 
0.075  per  cent,  or  perhaps  slightly  less,  and  at  the  highest  con- 
centration of  CuS04  employed,  namely  0.15  per  cent,  we  obtain 
an  increase  of  about  100  per  cent  of  nitrate  over  the  normal  soil 
to  which  no  copper  is  added.  That  such  remarkable  stimulation 
should  be  exercised  by  CuS04  for  the  nitrifying  flora  and  at  such 
high  concentrations  of  that  supposedly  toxic  salt  is  a  new  and 
interesting  fact  of  great  significance. 

While  our  results  agree  with  those  of  Fred  above  cited  as 
regards  the  lower  concentration  of  CuS04  which  he  employed, 
namely  0.01  per  cent,  they  are  wholly  at  variance  with  his  results 
as  regards  his  only  other  concentration,  0.1  per  cent  CuS04. 
The  latter  gave  Fred  no  stimulation  for  the  nitrifying  flora  what- 
ever, but  it  yields  us  nearly  the  maximum  point  of  stimulation 
of  CuS04  for  the  flora  in  question,  and  in  a  series  of  results  in 
which  the  existence  of  stimulation  is  throughout  remarkable. 
To  what  circumstance  these  differences  between  our  work  and 
that  of  Fred  are  attributable  is  not  readily  determined.  It  can 
scarcely  be  due  to  the  difference  in  the  strain  or  vigor  of  the 
nitrifying  flora,  for  we  have  obtained  similar  results  to  those 
above  described  with  widely  different  soils  which  were  employed 
in  corresponding  vegetation  experiments.  That  the  nitrifiable 
materials  were  wholly  different  may,  however,  be  a  factor  of  con- 
siderable significance.  Not  only  because  of  its  totally  different 
physical  nature,  but  because  of  possible  antagonistic  effects  which 
it  might  induce,  (NH4)2S04  might  well  be  expected  to  yield 
different  results  in  nitrification  work  from  those  obtained  with 
dried  blood  or  similar  forms  of  nitrogen,  especially  when  salt 
effects  are  studied. 


1914]       Lipman-Burgess:  Ammonification  and  Nitrification  in  Soils  135 

A  question  more  difficult  of  solution  is  that  involving  the 
cause  or  causes  of  the  stimulating  effects  of  CuS04  on  the 
nitrifying  flora  or  the  production  of  nitrates  in  soils.  The 
experiments  of  Fred  have  included  such  as  aimed  at  the  dis- 
covery of  the  reply  to  the  question  just  raised.  The  results  of 
such  experiments,  however,  would  seem  to  throw  no  positive 
light  on  its  solution.  Two  factors  do  seem,  nevertheless,  to  have 
been  eliminated.  But  little  support  was  found  for  the  idea  that 
poisons  like  the  salts  above  employed  destroy  soil  toxins  which 
might  interfere  with  bacterial  efficiency,  as  well  as  for  the  further 
idea  that  the  effect  of  such  poisons  in  the  destruction  of  amoebae 
might  manifest  itself  by  a  stimulation  for  the  soil  organisms. 
It  seems,  to  use  Fred's  expression,  to  be  a  "Reizwirkung"  on 
the  part  of  the  salts  and  other  poisons  added  to  the  nitrifying 
flora,  but  the  question  as  to  how  this  "  Reizwirkung "  is  accom- 
plished still  remains  unanswered.  This  question  is  further  dis- 
cussed below. 

ZnS04 

Zinc  evidently  exercises  relatively  the  same  influence  on  the 
nitrifying  flora  as  copper.  Like  the  latter,  it  stimulates  but  very 
slightly  at  a  concentration  of  0.0125  per  cent  ZnS04,  does  so 
very  definitely  at  a  concentration  of  0.025  per  cent,  very 
markedly  at  0.05  per  cent,  and  still  more  markedly  at  concen- 
trations of  0.075  per  cent  and  0.10  per  cent,  at  which  latter  the 
highest  point  of  stimulation  is  reached.  Concentrations  in  excess 
of  0.1  per  cent  ZnS04  seems  to  be  much  less  stimulating  to  nitrate 
production  than  similar  amounts  of  CuS04.  The  absolute 
amounts  of  nitrate  produced  by  the  nitrifying  flora  under  the 
stimulus  of  zinc  salts  seem  to  be  throughout  slightly  smaller  than 
those  produced  by  similar  flora  in  the  presence  of  CuS04. 

FeS04 

With  FeS04  we  obtain  effects  on  the  nitrifying  flora  which 
possess  many  characteristics  different  from  those  obtained  with 
CuS04  and  ZnS04.  In  the  first  place,  stimulation  is  marked 
with  the  smallest  concentration,  0.0125  per  cent  FeS04,  while 
it  is  either  extremely  slight  or  wanting  with  the  other  salts  at 


136         University  of  California  Publications  in  Agricultural  Sciences     [Vol.  1 

similar  concentrations.  While  stimulation  continues  up  to  and 
including  concentrations  of  0.075  per  cent,  it  is  not  so  great  as 
with  the  smallest  concentration,  and  increases  or  decreases  with 
the  different  amounts  of  FeS04  employed,  with  no  regularity. 
Then,  beyond  the  concentration  of  0.075  per  cent  FeS04,  a 
wholly  inexplicable  sudden  toxicity  manifests  itself,  and  stranger 
still,  beyond  that  point  with  two  additional  and  larger  concen- 
trations employed  we  obtain  the  most  marked  stimulation  in  the 
whole  series  of  cultures  and  about  equivalent  to  the  stimulation 
effected  by  CuS04  at  similar  concentrations. 

The  causes  of  this  erratic  behavior  of  FeS04  are  a  mystery 
as  yet.  We  suggest  that  the  rapidity  of  oxidation  of  the  FeS04 
may  vary  and  so  the  ferrous  and  ferric  salts  may  yield  different 
effects,  though  this  explanation  does  not  appear  wholly  satis- 
factory. The  distribution  of  the  salt  in  the  soil  culture  may  also 
be  a  factor.  What  appears  to  us  as  another  promising  explana- 
tion for  the  behavior  of  the  FeS04  under  discussion  is  its  effect 
on  the  physical  condition  of  the  soil  and  the  modification  of  the 
latter 's  absorptive  power  for  moisture  and  gases.  Whether  or 
not  the  effects  noted  may  be  related  to  transformations  in  the 
amounts  of  available  bacterial  foods  present  cannot  be  answered 
from  the  data  in  hand. 

PbS04 

In  this  series  of  cultures  we  are  confronted  with  effects  which 
are  in  certain  definite  ways  different  from  either  those  of  CuS04 
and  ZnS04,  on  the  one  hand,  and  FeS04  on  the  other  hand. 
Lead  is  the  only  one  of  the  metals  used  above  which  exercises  an 
unquestionable  and  marked  toxicity  on  the  nitrifying  flora  at 
even  the  lowest  concentration  (0.0125  per  cent  PbS04).  But 
while  that  is  so,  larger  amounts  of  PbS04  make  conditions  in  the 
soil  more  favorable  for  nitrification  as  the  lead  salt  increases 
in  concentration  from  0.0125  per  cent  to  0.05  per  cent,  at  which 
point  nitrification  is  about  as  active  as  in  the  normal  soil.  When, 
however,  the  PbS04  is  still  further  increased  we  obtain  what 
appears  to  be  a  definite  stimulation  to  nitrification.  Oddly 
enough,   however,   at  a   concentration   of  0.1    per   cent,   PbS04 


1914]       Lipman-Burgess :  Ammonification  and  Nitrification  in  Soils  137 

behaves  like  a  similar  concentration  of  FeS04  to  the  nitrifying 
flora  and  becomes  about  as  toxic  as  0.0125  per  cent  was  shown 
to  be  above.  Then  at  0.0125  per  cent  PbS04  the  latter  again 
behaves  like  FeS04  in  that  definite  stimulation  follows  the  tox- 
icity of  the  previous  concentration,  but  while  the  stimulation  of 
the  FeS04  at  a  similar  concentration  is  very  marked  it  is  only 
slight  in  the  case  of  PbS04.  Then,  again,  at  the  further  con- 
centration of  0.15  per  cent  PbS04,  the  latter  acts  entirely  differ- 
ently from  all  the  other  salts  in  that  it  becomes  markedly  toxic 
again. 

The  idiosyncracies  of  PbS04  as  regards  the  nitrifying  flora 
of  a  soil  are  even  more  numerous  than  those  of  FeS04.  How- 
ever, it  must  be  borne  in  mind  that  PbS04  is  a  very  insoluble 
salt  and  while  the  other  salts  are  capable  of  being  added  in 
solution  to  the  cultures,  PbS04  has  to  be  mixed  with  the  soil  as 
thoroughly  as  possible  in  its  dry  state.  This  fact  would  account 
for  a  poor  distribution  of  the  salt  through  the  soil  mass  and 
thus  indicate  one  possible  cause  of  the  extremely  erratic  behavior 
of  PbS04  in  nitrification  cultures.  The  fact,  however,  that 
FeS04  and  PbS04  behave  alike  at  a  concentration  of  0.1  per 
cent  would  seem  to  be  more  than  a  mere  coincidence,  and  some 
of  the  speculations  made  above  with  respect  to  FeS04  and  its 
behavior  may  have  some  cogency  here.  The  most  striking  fact, 
however,  gleaned  from  the  series  of  cultures  with  PbS04  is  that 
the  latter  possesses  a  stimulating  power  toward  the  nitrifying 
flora.  Considering  the  well-known  toxic  powers  of  lead  with 
respect  to  other  organisms,  this  fact  assumes  considerable  im- 
portance. In  all  cases,  of  course,  the  stimulating  powers  of  the 
other  salts  are  much  greater  than  that  of  PbS04. 

General  Discussion 

The  experimental  data  above  submitted  constitute  the  first 
series  of  systematic  experiments  dealing  with  the  effects  of  copper, 
zinc,  iron  and  lead  on  the  ammonifying  and  nitrifying  flora  of 
the  same  soil.  Once  more  we  are  confronted  by  the  striking 
differences  between  the  physiological  characteristics  of  the 
ammonifying  and  the  nitrifying  flora  of  soils.     Other  similar 


138         University  of  California  Publications  in  Agricultural  Sciences     [Vol.  1 

cases,  though  not  so  marked,  have  been  commented  on  elsewhere 
by  one  of  us  (see  literature  above  cited).  While  the  sulfates 
of  all  the  metals  named  are  toxic  to  ammonification,  and  in  very 
low  as  wrell  as  very  high  concentrations,  ranging  from  50  parts 
per  million  to  2500  parts  per  million,  they  manifest  a  relatively 
low  toxicity  for  the  flora  concerned  in  that  phase  of  nitrogen 
transformation.  On  the  other  hand,  all  the  same  metals  exercise, 
in  a  large  variety  of  concentrations,  a  marked  stimulating  effect 
on  the  nitrifying  flora.  That  differences  of  such  magnitude 
should  exist  between  two  flora  of  the  soil,  one  being  presumably 
dependent  for  its  raw  material  on  the  other,  is,  to  say  the  least, 
amazing  and  not  wholly  explicable.  Since,  however,  nitrification 
in  the  broader  sense  of  the  term  represents  the  algebraic  sum  of 
the  activities  of  all  forms  of  nitrogen-transforming  bacteria  in 
the  soil,  it  would  appear  from  our  results  that  the  net  effect  of 
the  activity  of  the  metallic  salts  tested  is  to  insure  a  larger 
nitrate  supply  in  the  soil.  This  must  be  so,  since  in  the  most 
extreme  cases  above  studied  ammonification  is  depressed  only 
to  an  extent  which  lowers  the  total  ammonia  yields  below  that 
of  the  salt-free  soil  by  about  30  per  cent,  whereas  nitrate  pro- 
duction is  frequently  enhanced  in  efficiency  by  the  presence  of 
the  same  metals  to  an  extent  which  doubles  the  yield  of  a  normal 
soil  flora. 

Our  findings  would  therefore  render  much  easier  the  explana- 
tion of  the  oft-noted  stimulating  effect  of  copper,  in  particular, 
and  metallic  salts  in  general  (in  proper  concentrations),  on  the 
higher  plants,  regarding  which,  also,  we  have  accumulated  con- 
siderable data.  If  a  larger  nitrate  production  in  the  soil  follows 
the  application  of  a  metallic  salt  as  above  shown,  the  nitrogen 
nutrition  of  plants  must  go  on  with  greater  facility  and  adequacy ; 
hence  increased  growth. 

As  above  intimated,  the  underlying  causes  of  our  very  inter- 
esting results  are  not  easy  to  discover.  In  addition,  however,  to 
the  speculations  on  that  subject  which  we  make,  and  which  we 
recognize  for  more  reasons  than  one  as  far  from  satisfying,  we 
have  obtained  some  experimental  evidence  on  the  point  in  ques- 
tion which  possesses  more  cogency.  It  appears  from  these  that 
water  absorption  is  hastened  by  germinating  seeds  and  young 


1914]       Lipman-Burgess :  Ammonification  and  Nitrification  in  Soils  139 

plants  in  the  soil  in  the  presence  of  copper.  Why  might  not  a 
similar  effect  be  exercised  by  metals  as  regards  the  nitrifying 
bacteria  which,  physiologically  speaking,  in  every  way  resemble 
the  higher  plants  much  more  than  the  other  soil  flora  ? 


Conclusions 

1.  Copper,  zinc,  iron  and  lead  exercise  toxic  effects  on  the 
ammonifying  flora  of  a  sandy  soil  from  Southern  California  in 
all  concentrations,  ranging  from  50  to  2500  parts  per  million  of 
their  sulfates. 

2.  Such  toxicity  is  relatively  small,  however,  and  is  more 
marked  at  concentrations  below  0.1  per  cent  than  above  it,  in 
most  cases. 

3.  The  metals  named  exercise  no  stimulating  effect  on  the 
ammonifying  flora  at  any  concentration. 

4.  Copper,  zinc,  iron  and  lead  exercise  marked  stimulating 
effects  on  the  nitrifying  flora  of  the  same  soil  and  frequently 
more  than  double  the  normal  nitrate  yield. 

5.  The  same  metals  may  in  very  small  concentrations  exercise 
toxic  effects  on  the  nitrifying  flora  or  no  effect  at  all,  but  they 
are  markedly  stimulating  at  much  higher  concentrations. 

6.  With  the  one  exception  of  lead  sulfate,  the  metals  named 
showed  very  marked  stimulation  for  nitrification  even  at  0.15 
per  cent,  the  highest  concentration  employed. 

7.  Comparisons  are  given  of  our  work  with  that  of  one  or 
two  other  investigators  on  portions  of  the  same  subject.  In  some 
cases  our  results  confirm  previous  work.  In  other  cases  they  are 
wholly  at  variance  with  it. 

8.  A  brief  discussion  is  given  of  possible  causes  for  the  effects 
of  the  metallic  salts  under  consideration  on  the  soil  flora. 

9.  Fuller  consideration  will  be  given  in  another  paper  to  the 
theoretical  phases  of  the  subject  treated  above. 

Transmitted  March  6,  1914. 


